ÁREA: Bioquímica e Biotecnologia
TÍTULO: OPTIMIZATION OF ETHANOL PRODUCTION BY YEAST STRAIN 63M AND A STRATEGY TO IMPROVE CELL SURVIVAL AT HIGH TEMPERATURES
AUTORES: SOUZA, C. S. (ICB-USP) ; OLIVEIRA, K. F. (ICB-USP) ; CAPELA, M. V. (IQ-UNESP) ; TOGNOLLI, J. O. (IQ-UNESP) ; LALUCE, C. (IQ-UNESP)
RESUMO: The effects of process conditions on viability and ethanol formation were analyzed in synthetic medium via response surface methodology, using a 23 full central composite design. Aiming at retaining a maximal viability and a maximal ethanol produced at the end of the fermentation, the MINITAB 14.0 gave the following optimized values: 30°C, initial sucrose concentration of 200 g/L and inoculum of 40 g/L. Under such conditions, the viability was 98.8% and the ethanol concentration was 80.8 g/L. Above 30°C, the minimization of the effects of increasing temperatures on viability and ethanol production was studied in pulse fed-batch cultures. The lethal effect of ethanol on cell survival was overcome by reducing the sucrose concentration in the fermentation system at high temperatures.
PALAVRAS CHAVES: saccharomyces; ethanol production; high temperatures
INTRODUÇÃO: Classical fermentations (bioethanol, beer, wine, sake, distilled spirits, baker’s yeast) are usually carried out using strains of Saccharomyces cerevisiae as starter. The use of high sugar concentrations and high cell densities reduce the fermentation time. Fed-batch cultivations have successfully been used in fuel-ethanol production. Despite the achievements in the field of the ethanol production, halted or sluggish fermentations still persist as a lasting threat for 6 to 7 months of operation in an ethanol plant. In fact, temperature is one of the main causes of interrupted or a slower fermentation process in warm regions of tropical countries where high temperatures above 34°C can be reached within the reactors. Optimizing an industrial process with respect to ethanol concentration, productivity and yield require the quantification of the dynamic behaviour of the yeast population at very high ethanol concentrations and this is not a simple task under continuous culture conditions (ALFENORE et al., 2004). It is easier to define optimal monitoring strategies for a fermentation process, when the extreme limits of the yeast’s tolerance to ethanol and temperature are previously determined. The aim of the present work was to determine the limits of the yeast’s tolerance to high temperatures and sugar concentrations at high cell density.
MATERIAL E MÉTODOS: Yeast strain: strain 63M (LALUCE et al., 2002).
Synthetic medium: The medium used was that described by THOMAS et al. (1998) containing sucrose as carbon source (100-200g/L). Variations in the sucrose concentration were made: 100-200g/L.
Inoculum propagation: overnight growth on the synthetic medium containing 10% sucrose and 2% yeast extract.
Experimental design method and statistical analysis involving the following: a) Response surface design: 2^3 full central composite design; b) Independent variables were: temperature (30-40°C), initial sucrose (100g/L-200g/L sucrose) and inoculum concentrations (30-40g/L in dry weight); c) Dependent responses were: ethanol and viability; d) Software: MINITAB 14 and STATISTICA 6.0; e) Experiment carried out in agitated (100 rpm) batch cultures as follows: portions of 100mL synthetic medium at pH 4.5 were propagated in 250mL Erlenmeyer flasks
Fed-batch fermentations: a 200 mL mini-reactor was operated as follows: a) addition of a 2-fold concentrated synthetic medium without sucrose (50 mL) at pH 4.5 and concentrated yeast cream (4 g/25 mL, dry weight); b) addition of decreasing pulses of sugar solution (800g/L), every 30 min within the first two-hour period of fermentation to obtain sucrose concentrations (100-200g/L) as required by the experiments; c) addition of NaOH solution (2.4 mol/L) immediately after the addition of the first sucrose pulse to raise the pH to 4.5; e) temperature (30-40°C) and the final working volume was 100mL.
Analytical methods: a) Cell viability was determined using the methylene blue method (LEE et al., 1981); b) residual sugar using the 3,5-dinitrosalicylic acid method (MILLER, 1959); c) ethanol using a gas chromatograph; c) cell biomass was washed and dried at 105°C until constant weight.
RESULTADOS E DISCUSSÃO: The analyses of the results obtained using the 2^3 full central composite design showed a quadratic model of ethanol production and linear model for the viability. The response surface plots showed that the maximal viability values were dependent on sucrose concentrations and temperatures but not dependent on the inoculum size. High ethanol yields were dependent on temperatures, sucrose and inoculum concentrations. The optimized values for the variables in the batch culture were: 30°C, initial sucrose concentration of 200g/L and inoculum of 40g/L. Under the aforementioned conditions, the value of viability was 98.8% and the ethanol was 80.8g/L. Experiments were then carried out in pulse fed-batch cultures in order to minimize the effects of temperatures greater than 30°C. At 37°C, a high retention of viability was obtained at the end of fermentation by adding a total dose of sucrose equivalent to 150g/L. The lower values of viability at sucrose concentrations above 150g/L seem to be due to a fast accumulation of internal ethanol over short fermentation periods. In a report from literature, longer fermentation periods were obtained when temperatures decrease within the reactor (JONES & INGLEDEW 1994). Despite the fast sugar uptake shown by strain 63M at low sucrose concentrations (up to 150g/L), high values of final viability were obtained due to the use of a high cell density. It has been described that the maintenance of viability during fermentation is favored by a high cell density (UENO et al. 2002). At 40°C, the concentration of added sucrose had to be reduced to 100g/L in order to maintain a high viability. The low values of final viability indicated that it is not possible to run successive fermentations using 200g/L sucrose with cell reuse above 37°C.
CONCLUSÕES: Conditions resulting from the experimental validation were: 80.8g/L ethanol (20.2g/L/h); 98.8% viability; constant values of residual sugar and ethanol yields after 7h fermentation at 30°C. Above 30°C, the sugar concentration has to be decreased in order to minimize the effects of high ethanol levels on viability. At 37°C and with a sucrose concentration up to 150g/L drops in viability were obtained with a productivity of 25.4g/L/h. At 40°C and with a sucrose concentration up to 100g/L the losses in viability would decrease temporarily until temperature control is restored within the reactor.
AGRADECIMENTOS: The authors are grateful by FAPESP (proc. 2005/01498-6) and CAPES.
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